DESCRIPTION: This proposal is divided into two parts designed to identify and characterize active Tourist/stowaway elements and to study mechanisms of regulation related to retrotransposons. Whether the Tourist element represents an LTR type sequence from a retrotransposon or a DNA transposable element is not known. Two subfamilies of Tourist elements (B2 and Hbr) have been identified of which representative elements appeared to have transposed recently. All members are less than 350 bp and do not have extensive ORFs. A necessarily important objective is to identify active elements and to discern the mode of transposition of this class of elements. The structurally similar Stowaway elements may then have a similar mechanism of transposition. The PI recognizes that identification of an active element is not a trivial undertaking. One approach will be to use a collection of DNA pools representing 40,000 Mu containing individual maize plants and PCR with pairs of primers one of which is Tourist subfamily specific and one of which is anchored to a known gene. The products of this amplification will be probed for evidence of the presence of the target genes in order to avoid isolation of random amplifications of Tourist DNA. Whether Tourist elements are DNA or RNA based may be distinguished by whether excision from DNA occurs. Southern blot hybridization will be used in order to test for evidence of Hbr excision from hm1::dHbr-1062 containing plants where it was discovered. In addition, reverse transcription PCR will be performed in order to determine whether Hbr is spliced from the RNA and therefore may represent a retrotransposon of the R2Bm insect type. Sequence analysis of 20 to 50 members will be undertaken in order to identify features of the element which appear to be conserved. This may be helpful in the identification of "intact" elements. Restriction sites may be identified withwhich full length elements flanked by Tourist sequences can be identified. Some transposable elements are mobilized by stress. Whether long term selective breeding results in amplification of Tourist elements will be determined by genomic analysis of strains provided from the Ilinois Long Term Selection Exeriment covering 96 cycles of selection for high/low protein and oil traits and one population which after 48 cycles was subjected to reverse selection. Interestingly Hbr elements have been identified in the flanking regions of genes so that the element occurs in the 3' nontranslated region. The hm1 transcript containing the Hbr insertion is present at lower levels than the transcript from wt cells. The PI proposes to test the hypothesis that these elements can destabilize target 3' sequences by first determining whether the rate of transcription of the hm1 gene can account for the known reduced level of expression of the mutant gene. Other cDNAs will be isolated from genes associated with Hbr and tested indirectly for evidence of accelerated turnover. If evidence is gained that regulation may be at the level of turnover, then a reporter system will be set up with which to monitor the effect of Hbr sequences on RNA stability. Plant transposable elements have been shown to be capable of transposition in heterologous species and this approach offers the advantage, in the proper context, of low background. Rice has Tourist elements and so presumably also any associated functions, but does not have the Hbr or B2 subfamilies. Hbr and B2 will be introduced and the plants monitored for evidence of expansion. One concerning property of maize retrotransposons from the point of view of the experimentalist is the absence of transcripts. The PI hypothesizes that this may reflect host restriction mechanisms such as are observed in trans-inactivation and in silencing where amplification of gene number is accompanied by down regulation of expression. One novel and recently described mechanism of such regulation is pathogen- derived resistance in which case it appears that prior exposure to RNAs for viral coat proteins can upon reexposure trigger specific turnover of this RNA. This may be mediated by generation of a double stranded RNA by host cell polymerases; such RNAs could turnover rapidly. Nuclear run-off assays will be used in order to determine whether degradation of retrotransposon RNAs is a possible explanation for low steady state levels of RNA. The Hopscotch element which is represented at low copy number will be used for the first of these experiments. An interesting discovery of the previous funding period was the presence of a gag fusion to 740 codons of the cellular gene for an H+ ATPase carried by a reverse transcriptase defective Bs1 element. Despite a number of nucleotide changes relative to the cellular gene, the ORF is preserved, suggesting that it may serve some selective advantage to the host or the Bs1 element. Since it is not possible to monitor Bs1 transposition, effects of expression of the gag-pma fusion by identifying plants which lack this Bs1 element and scoring them for differences with sibs which do have the element. If significant differences are surfaced, then the effect of overexpressing the gag-pma fusion in Arabidopsis will be determined.